Project Summary Congenital heart disease (CHD) leads to severe morbidity and mortality to children in the US and worldwide. Despite this impact on child health, we simply do not understand the genetic causes of CHD. Recently, trio based whole exome sequencing has identified a class of voltage-gated potassium channels (multiple KCNH family members) as candidates for CHD and, specifically heterotaxy, a disorder of left-right (LR) patterning that has a severe effect on cardiac function. However, a molecular role connecting potassium channels to structural heart disease and heterotaxy is unprecedented. We propose, and our preliminary data support, that KCNH6 defines a new paradigm for cell signaling in early embryonic cells. Our data support an electrophysiological model where specific germ layers fates (paraxial mesoderm and ectoderm) are dependent on an ion channel network. Our overarching hypothesis is that K+ channels define electrical membrane potential and regulate voltage gated Ca2+ channels that establish an exit from pluripotency towards specific cell fates, gastrulation, and LR patterning providing a plausible mechanism for our patients with Htx and CHD. Our electrophysiological pathway then integrates with biochemical signaling pathways that define specific cell fates in the embryo. In this proposal revision, we will focus on KCNH6 to see if gene depletion leads to LR patterning defects in Xenopus. In addition, we will test where in the LR patterning cascade, KCNH6 plays a role. Then, using a series of judiciously chosen chemical and ionic perturbations, we will test if membrane potential is indeed essential for pluripotency, cell fate, and calcium regulation. Due to the novelty of this project, we will also perform unbiased genomics (RNAseq) for discovery of transcriptional targets of ? Vm. Finally, we will measure electrical properties electrophysiologically using both whole-cell voltage clamp and intracellular recordings and determine the various currents that define membrane potential in early germ cells. A major strength of our proposal is our expertise; we have forged a collaboration between Xenopus developmental biologists and electrophysiologists that will allow us to rigorously investigate membrane potential as an embryonic patterning mechanism.